Methods of DNA assembly

RMDAP contains donor and recipient vectors, and uses bacterial strain SW106 as the recipient host, which has all the requisite in vivo recombination proteins, including an arabinose-inducible cre gene and a defective λ prophage with recombination proteins exo, bet, and gam controlled by the temperature-sensitive repressor cI857 [17]. The assembly of DNA fragments was carried out by Cre/loxP-mediated and Red-mediated recombination events alternately and circularly.

First, two original donor vectors, pOSB100 and pOSB200, were constructed (Figure 1A) containing the sequences “loxP+E1+MCS+E2” and “loxP+E2+MCS+E1” respectively (E1 = rare restriction enzyme set 1, MCS = multiple cloning site, E2 = rare restriction enzyme set 2). We chose the pMD18-T simple vector (Takara) as a skeleton with ampicillin resistance, and MCS from pBluescript SKII. Rare restriction enzyme set 1 (E1) contains AscI, SbfI, and homing endonuclease I-PpoI sites, while rare restriction enzyme set 2 (E2) contains PacI, PmeI, and homing endonuclease I-SceI sites. The homing endonucleases are double-stranded DNases with large, asymmetric recognition sites (12–40 bp) (New England BioLabs), so recognition sites are extremely rare. As PacI, PmeI, AscI, and SbfI are the restriction enzymes able to identify eight nucleotide sequences, the occurrence of these is also relatively low, and so they could been carried out identification of the vector by enzyme digestion, and if necessary, also excise the intermediate vector's backbone. All satellite vectors later described are derived from these two original vectors. The RMDAP inherits all of the advantages of the former system [11], [16], but also has greatly expanded functional and utilization aspects. These methods are completely compatible.

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Figure 1. Physical maps of original donor vectors and recipient vectors carrying different marker genes.

(A) Schematic illustration of the two original entry vectors pOSB100 and pOSB200, from which the series is derived. The fragment introduced into the pMD18-T simple vector contained a loxP site (blue arrow), a multicloning site (red box), and two rare restriction enzyme sets E1/E2 (underlined). These two sets are in opposite orientation. Ori, pUC replicon; Amp^r, ampicillin resistance gene. (B) Construction of binary transformation vectors equipped with no plant selection gene, for marker-free type assembly, or the commonly used plant selection genes of hptII and bar, based on the pCAMBIA family. LB and RB, left border and right border of the T-DNA; HPTII, hygromycin resistance gene cassette; BAR, phosphinothricin resistance gene cassette.

https://doi.org/10.1371/journal.pone.0019883.g001

Based on the backbone of vector pCAMBIA (CAMBIA, Canberra, ACT, Australia), we designed and constructed a set of three T-DNA destination vectors for various tasks, carrying no plant selectable marker gene or genes encoding resistance to hygromycin (hptII, hygromycin phosphotransferase) and phosphinothricin (bar, phosphoinothricin acetyltransferase). The component containing loxP and E1 was cloned and introduced into corresponding sites of pCambia1300 and pCambia3301, producing pDES100, pDES200 (HPTII), and pDES300 (BAR) (Figure 1B). In particular, destination vector pDES100 was used to construct a maker-free binary vector, combined with satellite plasmid containing a plant selection gene that could be induced to allow removal.

Before assembling the DNA fragments, according to the specific purpose of the experiment, we need to choose appropriate satellite vectors. The assembly of the first two genes is shown schematically in Figure 2A. We adopted in vivo Cre/loxP-mediated recombination via loxP to allow the donor vector pOSB1xx to co-integrate with the recipient vector. A mixture of donor vector and recipient vector was electroporated together into E. coli strain SW106, which had been induced expression of the cre gene by adding filter-sterilized arabinose. This formed a vector mixture containing the co-integrated vector and parent vectors in the same strain. To create pure clonal populations of the recombinant, it was necessary to retransform it to E. coli DH5α at a concentration of less than one molecule per cell. The resulting co-integrative plasmids were mini-preped and purified to be digested completely by the homing endonuclease to remove the backbone. Next (Figure 2B), we performed Red-mediated recombination to retrieve another fragment. A pair of primers, each with at least 50 base homologies at the 5′-ends corresponding to the flanking sequence of the homing endonuclease site, and about 20 bases of the second gene or functional fragment were designed to amplify a target DNA fragment through high-fidelity Taq DNA polymerase. The resulting gene segment was ligated to the TA-cloning vector, followed by sequencing. The correct plasmid was transformed into strain SW106 to prepare competent cells after induction of the λ recombination genes. Finally, the linear vector fragment excluding the skeleton of the donor plasmid was transformed into the SW106 cells to achieve recombineering, and then screened for the correct clone on the right medium. At this point, two genes of the first round were integrated into the expression vector. For the second round of gene integrations, we chose pOSB2xx as the donor vector. In short, heterologous fragments could been stacked into the expression vector alternately according to the above procedures, in only a few days, using simple molecular biology operations.

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Figure 2. Schematic diagram for the round of gene stacking of the first two genes.

(A) Through in vivo Cre/loxP-mediated recombination, the donor plasmid with the first gene was integrated into the recipient vector via loxP. Then the backbone of donor plasmid was excised to form a linear targeting construct. Next, the second gene or DNA fragment was retrieved by in vivo Red-mediated recombination and gap repair. (B) Detailed schematic illustration of how Red-mediated recombination might occur. E1/2, rare restriction enzyme set 1 / 2; Ori, pUC replicon; Amp^r, ampicillin resistance gene; Kan, kanamycin selection gene; pVS1, pVS1 replicon; M, marker gene; LB and RB, left border and right border of the T-DNA.

https://doi.org/10.1371/journal.pone.0019883.g002

Features of the modular vector

Functional analysis for crop improvement can be approached in a number of ways, most of which rely on overexpression of transgenes, RNAi of endogenous genes to manipulate biological processes in transgenic plants, and marker-free vectors, related to the safety of genetically modified organisms (GMO). Therefore, the specific design of the satellite vectors is based on these three aspects: overexpression vector with different promoters, RNA interference vector, and marker-free vector.

Gain-of-function can be achieved by placing a gene of interest under the transcriptional control of a constitutive promoter. When carrying out multigene transformations, adoption of the homologous promoter has been found to creat certain transgene-silencing effect [18], making different promoters of the expression cassette necessary. Thus, to design modular vectors for constitutive ectopic expression, we developed a set of six couples of modular plasmids (Figure 3A), each of which carried a different pair of promoter and terminator sequences: cauliflower mosaic virus 35S promoter (p35S), nopaline synthase gene promoter (pNOS), octopine synthase promoter (pOCS), manopine synthase promoter (pMAS), tobacco cryptic promoter (pENTCUP2) strong, constitutive promoters constructed for dicot plants, and maize ubiquitin promoter (pUBI) for monocot plant transformation. Besides these, another chloroplast-targeted satellite vector was also constructed successfully (Figure S1). Meanwhile, because the production of the constructs is laborious and often hampered by inappropriately positioned restriction sites, we also adopted Gateway cloning technology to join fragments in a predefined order, orientation, and reading frame [19]. Due to the presence of accessible restriction sites, we can further extend the family of plasmids to carry various promoters and terminators, especially inducible or tissue-specific promoters, through traditional ligation to maintain the high flexibility of the system.

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Figure 3. Schematic representations of the pOSB modular vector series.

There are three functional types of modular vector containing 14 pairs of satellite vectors. In addition to special illustrations, pOSB2xx vectors can be digested using restriction enzymes PacI (PmeI) and AscI (Sbfi) to obtain the whole expression cassette, and inserted into pOSB100, resulting in pOSB1xx. (A) The gateway destination cassettes consist of R1 (attR1 recombination attachment site), ccdB (negative selection marker), and R2 (attR2 recombination attachment site) sequences, flanked by PacI, KpnI, XhoI and XbaI, NotI, SacII, SacI, allowing the replacement with different regulatory elements. All promoters and terminators (cauliflower mosaic virus 35S promoter(p35S), nopaline synthase gene promoter (pNOS), octopine synthase promoter (pOCS), manopine synthase promoter (pMAS), tobacco cryptic promoter (pENTCUP2), and maize ubiquitin promoter (pUBI)) are introduced into corresponding sites, producing a variety of constitutive ectopic gene expression vectors. The chloroplast-targeted tissue-specific expression vector was constructed through the ribulose 1,5-bisphosphate carboxylase small subunit of tobacco driven by promoter p35S. (B) Outline of the different RNAi vectors produced in this study. Two inverted repeats of the gateway destination cassettes containing attP or attR sites locate on both sides of the intron driven by promoter pCaMV35S promoter tetramer. (C) Three types of marker-free satellite vectors: an estrogen-inducible marker-free vector, an acetanilide -inducible marker-free vector and a general-purpose type of marker-free vector. Ori, pUC replicon; Amp^r, ampicillin resistance gene; ter, terminator; HPTII, hygromycin resistance gene cassette; NPTII, kanamycin resistance gene cassette; G10-90, a strong synthetic constitutive promoter; XVE, a chimeric transactivator containing the regulatory domain of an estrogen receptor; CRE, Cre recombinase with an intron.

https://doi.org/10.1371/journal.pone.0019883.g003

Long double-stranded RNAs, in particular hairpin RNAs (hpRNAs), can be used to trigger gene silencing by RNA interference, and have been used for highly effective post-transcriptional gene silencing in plant cell research [20]. Using vectors of pHellsgate proved to be a straightforward method for assembly by Gateway recombination of hpRNA constructs, designed for high-throughput gene silencing in plant cells [21]. Through the LR or BP reaction with attL1(P1)-trigger-attL2(P2), the trigger sequence is incorporated into both sides of the intron in opposite orientations. For the RNA-interfering modular satellite vector, we adopted this series of vectors with the NotI restriction site and succeeded in transferring the RNAi expression cassette to the intermediate vector (Figure 3B). Because the pHellsgate vectors allow production of an hpRNA construct in a one-step or two-step process, we also retained these two ways.

Although no reports describe the effects of selectable marker genes on human health or environmental safety, public concerns over the safety of transgenic plants cause fierce debate. Selectable marker gene facilitates genetic transformation of plants, but generally it is not required for expression of the trait gene in the transgenic plant, and may even be harmful. How to delete a selectable marker gene has become an important research topic in the application of transgenic research. The Cre/loxP recombination system of the E. coli bacteriophage is a commonly used technique, and we took advantage of the general applicability of this system in bacteria and plants, i.e., the assembly of several transgenes in bacteria and removal of the selectable marker gene in plants by the inducible promoter, to construct two chemically induced marker-free satellite vectors. When foreign genes are introduced into plants, it is possible to remove the marker gene in vivo using estrogen [22] or acetanilide [23]. Meanwhile, to meet the needs of genetic breeding, we also generate a general-purpose type of marker-free vector in which the selection marker genes hptII and nptII (neomycin phosphotransferase II) were preset. By virtue of Gateway recombination, we can rapidly replace any inducible or tissue-specific promoter (Figure 3C).

Construction of plant multigene transformation vectors

As proof-of-principle experiments to evaluate the application of this modular vector system for multigene delivery, two transformation vectors with different expression units for different aims of genetic improvement were developed. Several genes, including Atriplex hortensis betaine aldehyde dehydrogenase (badh) involved in the accumulation of glycine betaine giving tolerance to salt stress [24], isoflavone synthase (ifs) from Medicago truncatula that a key enzyme in the flavonoids/isoflavonoids pathway [25], a phosphinothricin resistance gene (bar) and two reporter genes, gus and gfp, were chosen for gene stacking.

The first round of in vivo Cre/loxP-mediated recombination reactions was performed between pDES200 and pOSB103-GUS. The correct clones containing the co-integrative plasmid (pDES200-GUS) were obtained through electroporation of SW106 and heat-shock transformation of E. coli DH5α, followed by extraction of the plasmid and complete digestion with the homing endonuclease I-SceI to form a linear retrieval plasmid. The second GFP cassette of pOSB102-GFP was amplified with a pair of chimeric primers (Table S3), and transformed to SW106 to prepare the electrocompetent cells. To increase the frequency of subcloning by gap-repair, relatively longer homology arms are recommended; hence the retrieval plasmid previously mentioned could retrieve this DNA fragment conveniently to constitute pDES200-GUS-GFP. By repeating the process described above for additional target genes, gene assembly was carried out to deliver an IFS cassette of pOSB206-IFS to pDES200-GUS-GFP, and then a BADH cassette from pOSB205-BADH. Finally, we obtained a construct pDES200-GUS-GFP-IFS-BADH carrying four different foreign genes, excluding the selectable marker gene HPTII cassette (Figure 4A). Restriction analyses of a series of recombinant constructs containing various numbers of genes are described in Figure 4C.

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Figure 4. Schematic representation of plant transformation vector.

(A) Schematic structures of plant transformation vector pDES200-GUS-GFP-IFS-BADH. The T-DNA with left and right borders (LB and RB) is indicated. The different expression units of IFS cassette, BADH cassette, GUS cassette and GFP cassette, including the HPTII cassette, are indicated by orange arrows. (B) The inducible marker-free multigene transformation vector pDES100-GUS-BAR-HPT-free contains two interested genes of gus and bar. The hptII gene is responsible for hygromycin resistance needed for transgenic event selection and flanked by two direct repeat loxP sites, which could be excised by the induction of estrogen. (C) Restriction analysis of recombinant constructs containing different numbers of expression cassettes with restriction enzymes PacI and AscI. The restriction enzymes sites (PacI and AscI) for identification of the vector are also indicated. P1–P4, primers used for PCR analysis. LB: left border; RB: right border. Marker, 1-kb DNA ladder.

https://doi.org/10.1371/journal.pone.0019883.g004

Another marker-free multigene transformation vector was constructed to further verify the feasibility of the method for linking multiple transgenes. Vectors pDES100 and pOSB103-GUS were co-integrated by in vivo Cre/loxP-mediated recombination, producing pDES100-GUS. Then, through gene retrieval, the BAR cassette of pOSB104-BAR was joined to pDES100-GUS, resulting in pDES100-GUS-BAR. After another round of recombination with pOSB211, the desired recombinants were selected as pDES100-GUS-BAR-HPT-free (Figure 4B). Furthermore, the skeleton of the intermediate vector, containing an ampicillin-resistance gene for bacterial selection and the ColE1 origin of replication, did not need to be removed, and can be used for plasmid rescue procedures after transformation. We also tested the stability of the co-integrative plasmid of pDES100-GUS-BAR-HPT-free containing two replicons in E. coli and Agrobacterium using restriction enzymes to confirm that there were no aberrations in size. If the backbone of pDonor with loxP was excised, it was necessary to add another loxP, as a selectable marker gene should be placed between two loxP sites in direct orientation.

Plant transformation and expression analysis

First we respectively test the effectiveness of each expression cassette. The constitutive overexpression vectors, containing gfp gene introduced by gateway recombination, were individually introduced into onion epidermal cells by particle bombardment. GFP was detectable, confirming the activity of each expression cassette on the satellite vector (Figure S2). Moreover, leaf epidermal protoplasts of tobacco transformed with plasmid pOSB108-TP-GFP for chloroplast-targeted were isolated as shown Figure S3.

To evaluate the feasibility of this multigene transformation system, the construct pDES200-GUS-GFP-IFS-BADH was introduced into tobacco and Arabidopsis. thaliana by Agrobacterium-mediated transformation. Genomic PCR on independent transformants was performed, and 15 independent lines of T0 tobacco transformants were obtained. Because mgfp5 is not the most highly-fluorescent GFP mutant, and would be masked by the high chlorophyll autofluorescence of leaf cells, so we chose roots to enable easy visualization of the GFP. All of the transgenic plants showed GFP fluorescence and constitutive in situ GUS expression (Figure 5A), despite differences in the expression levels. To check for the expression of other genes, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of transgenic plants was carried out for each expression unit on the T-DNA. The predicted cDNA fragments of all transgenes, that were linked in the same T-DNA region, could be amplified in all tested samples.

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Figure 5. Expression analysis of transgenic plants.

(A) Leaves of transgenic plants were taken for in situ histochemical staining of GUS activity and roots were imaged by fluorescent confocal microscopy. Only one of the randomly chosen transgenic plants is shown. WT, wild-type; T0, T0 generation of transgenic tobacco; T1, T1 generation of transgenic A.thaliana. (B) Expression analysis of the different transcription units of transgenic A.thaliana. Confirmation of transgene expression of gfp gene and gus gene activity is symbolized by +, and lack of transgene activity is symbolized by −. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of all gene cassettes (HPTII, IFS, BADH) on the T-DNA from the leaves of 12 transgenic A. thaliana.

https://doi.org/10.1371/journal.pone.0019883.g005

After screening on agar-solidified media containing Murashige and Skoog salts (MS agar) with hygromycin (30 mg/L), 26 putative A. thaliana T1 transgenic plants were initially generated. Apart from two transgenic T1 lines exhibited poor fertility, 24 independent T2 transgenic A. thaliana lines were performed for further gene expression analysis with RT-PCR. Similar results were obtained as with tobacco, we found that all transgenes linked on the T-DNA were coexpressed. (Figure 5B).

To verify the capability for creating multigene transgenic plant of conditional elimination of selection markers, the construct pDES100-GUS-BAR-HPT-free was also introduced into A. thaliana lines by Agrobacterium-mediated transformation. Sixteen A. thaliana transgenic lines (T1) were obtained and transferred onto the inductive medium. After β-estradiol-induction (5 µm) for two weeks, all independent lines were then transferred to soil, and T2 seeds were obtained as a pool. Then we carried out PCR analysis with primer pairs specific for the excised sequences and flanking nonexcised sequences to verify the recombination events. The P1–P2 (612 bp) and P3–P4 (1341 bp) fragments will be amplified respectively in non-recombinant T-DNA, whereas P1–P4 (1184 bp) fragments can be detected in recombinant T-DNA (Table S5). Only those lines with unique P1–P4 fragment could be considered as complete DNA excision. Among the tested 100 lines randomly selected from the pool, 13 lines show complete marker excision (Figure 6A). The PCR products of P1–P4 fragments amplified from three marker-free transgenic plants (T2) were sequenced and all these sequencing results were identical (Figure 6B), indicating that the DNA fragment containing marker gene between two loxP sites was removed precisely.

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Figure 6. Identification of Cre/loxP-mediated DNA recombination event.

(A) PCR analysis of genomic DNA prepared from uninduced T1 plant and recombinant T2 transgenic plants (lines1, 2 and 3) using primers P1/P2, P3/P4, and P1/P4. Primer pairs used for each PCR reaction are indicated on the top of each lane. The expected PCR products from different combinations of primer pairs are indicated on the right. No amplification was observed with the genomic DNA of wild-type plants (WT) using any of the three primer pairs. M, 1-kb DNA ladder. (B) Sequence confirmation of marker gene excision in the genome of transgenic T2 lines. Part of the sequence at excision site is exactly as predicted, indicating that the excision occurred and all DNA sequences, including hptII gene, between two loxP sites were excised, with only one intact loxP site left.

https://doi.org/10.1371/journal.pone.0019883.g006

In conclusion, these results indicated that all heterologous genes on the T-DNA could be efficiently delivered into the plant genome as a whole, and could be successfully coexpressed.

Vector construction and bacterial strain

Standard molecular biology procedures and DNA manipulations [34] were performed to make all the constructs. Details of vector construction are available in the Text S1 [35], [36], [37]. If necessary, the vector backbone was dephosphorylated by Alkaline Phosphatase (Promega) prior to ligation. The sequences of primers used are listed in Table S1. All vectors containing the ccdB gene were propagated in the E. coli strain DB3.1, which contains the gyrA462 mutation that enables propagation of Gateway vectors encoding the ccdB gene [35]. SW106 cells were maintained at 32°C and used as the host for DNA assembly and integration and DH5α cells were employed for other transformations.

These vectors can be used in a wide range of functional genomics projects in plants and will be distributed to the research community for noncommercial research purposes upon request.

Subcloning of target genes to the entry vectors

The bar gene of pCambia3301, the gfp gene of pCambia1302, the badh gene of Atriplex hortensis, and the ifs gene of Medicago were amplified with the primers listed in Table S2, and ligated to pDonor 221 by the BP clonase or pCR®8/GW/TOPO® vector according to the manufacturer's instructions (Invitrogen), resulting in pENTR-bar, pENTR-gfp, pENTR-badh, and pENTR-ifs, respectively. The pENTR-gus was directly purchased from Invitrogen.

Due to the design of the experiment, the pENTR vector was used to transfer DNA fragments to the destination modular vectors using LR clonase according to the manufacturer's instructions (Invitrogen). Finally, we obtained the satellite vectors pOSB102-GFP, pOSB103-GUS, pOSB104-BAR, pOSB205-BADH, and pOSB206-IFS.

Multiple rounds of recombination reactions

For Cre/loxP-mediated plasmid co-integration, plasmids pOSB and pDES were co-electroporated into electro-competent cells of E. coli strain SW106, in which the cre gene had been induced by adding arabinose. Through screening on LB plates with kanamycin (50 mg/L) and ampicillin (50 mg/L), the resulting plasmid population was obtained and introduced into E. coli strain DH5α at a very low concentration to obtain a pure clonal population. After another round of screening as described above, co-integrative clones were obtained, isolated (AxyPrep Plasmid Miniprep Kit, Axygen), and digested completely with homing endonuclease (New England BioLabs and Promega) to produce the linear targeting vector.

For recombineering-mediated gene retrieval, chimeric primers were precisely designed according to the flanking sequence of the homing endonuclease site of the linear targeting vector and the second DNA fragment. The fragment was amplified with Phusion High-Fidelity DNA Polymerase (New England BioLabs) and ligated to the pMD18-T simple vector (Takara). The resulting plasmid was transformed to E. coli SW106 to grow at 32°C, and then the cultures were transferred to 42°C for a 15-min induction of Red recombination functions to prepare the electrocompetent cells. Finally, electroporation of the linear targeting vector (300 ng) was performed using a Bio-Rad electroporator under the following conditions: 1.80 kV, 25 mF capacitance, and 200 Ω resistance. Cells were spread on plates with kanamycin(50 mg/L) to screen for the correct recombinant clones by restriction analysis [29].

Plant transformation and expression analysis

Plants of Arabidopsis (A. thaliana Col-0), N. tabacum cv. Xanthi and Nicotiana benthamiana were placed in a growth chamber (25°C under a 16-h-light/8-h-dark cycle). The constitutive ectopic overexpression vectors were used for transient expression either of onion epidermis via a gene gun (Bio-Rad) or of N. benthamiana leaves via Agro-infiltration [38].

The A. tumefaciens strain Eha105 was used to transform tobacco via the leaf disc system [39] and A. thaliana Columbia-0 via the floral dipping method [40]. Six- to 8-week-old N. tabacum cv. Xanthi plants were transformed and explants were selected on MS medium containing 20mg/L hygromycin (Amersham Biosciences). The successful insertion of the exogenous genes was confirmed by PCR with genomic DNA isolated from leaves of independent transformants as templates. Transformed A. thaliana T1 seeds were selected on germination medium plates supplemented with 30 mg/L hygromycin, and T1 seedlings were transferred on an inductive medium containing 5 µM of 17-β-estradiol for 2 weeks.

Plant tissues were viewed directly for fluorescence analysis under a Nikon ECLIPSE TE2000-E inverted fluorescence microscope equipped with a Nikon D-ECLIPSE C1 spectral confocal laser scanning system. The in situ histochemical GUS assays were conducted using 5-bromo-4-chloro-3-indolyl glucuronide (Amersham Biosciences) as substrate [41]. The leaf tissues were cleared of chlorophyll by applying 90%, 80%, and 70% ethanol solutions in three consecutive steps, and photographed using a stereomicroscope.

Total RNA was extracted from leaf tissue with Trizol reagent (Invitrogen) according to the manufacturer's recommendations and digested with DNase I (New England BioLabs) to remove genomic DNA. The first strand of cDNA was synthesized using 5 µg of total RNA as template with the Reverse Transcription System (Promega) in a 20 µl reaction volume. Primers for each gene and size of fragment are shown in Table S4.